Bottom Line:
PTE treatment resulted in a dose- and time-dependent decrease in the viability of A549 cells.DAPT (a gamma secretase inhibitor) and Notch1 siRNA prevented the induction of NICD and Hes1 activation by PTE treatment and sensitized the cells to PTE treatment.The down-regulation of Notch signaling also prevented the activation of pro-survival pathways (most notably the PI3K/Akt pathway) after PTE treatment.

ABSTRACTAlthough pterostilbene (PTE) has been shown to have potent antitumor activities against various cancer types, the molecular mechanisms of these activities remain unclear. In this study, we investigated the antitumor activity of PTE against human lung adenocarcinoma in vitro and in vivo and explored the role of the Notch1 signaling pathway in this process. PTE treatment resulted in a dose- and time-dependent decrease in the viability of A549 cells. Additionally, PTE exhibited strong antitumor activity, as evidenced not only by a reduced mitochondrial membrane potential (MMP) and a decreased intracellular glutathione content but also by increases in the apoptotic index and the level of reactive oxygen species (ROS). Furthermore, PTE treatment induced the activation of the Notch1 Intracellular Domain (NICD) protein and activated Hes1. DAPT (a gamma secretase inhibitor) and Notch1 siRNA prevented the induction of NICD and Hes1 activation by PTE treatment and sensitized the cells to PTE treatment. The down-regulation of Notch signaling also prevented the activation of pro-survival pathways (most notably the PI3K/Akt pathway) after PTE treatment. In summary, lung adenocarcinoma cells may enhance Notch1 activation as a protective mechanism in response to PTE treatment. Combining a gamma secretase inhibitor with PTE treatment may represent a novel approach for treating lung adenocarcinoma by inhibiting the survival pathways of cancer cells.

Mentions:
The Western blot and immunofluorescence results showed that PTE increased the NICD protein level in A549 cells (Figure 4A and 4B). We then determined if the increase in the NICD protein level could augment Notch1 signaling by measuring the activity of a downstream target, Hes1. As observed for the NICD protein, PTE induced Hes1 expression (Figure 4A and 4C). We then examined the possible mechanism underlying the induction of NICD expression by PTE treatment. NICD is produced when the Notch1 receptor is cleaved by the gamma secretase complex, which is composed of four subunits (Presenilin-1, Nicastrin, anterior pharynx-defective 1 (Aph-1) and presenilin enhancer 2 (Pen-2)) [11], [12]. Therefore, PTE treatment induced NICD protein expression at least partially by increasing the gamma secretase activity. The treatment of A549 cells with PTE induced the expression of two of the subunits, Presenilin-1 and Nicastrin (Figure 4A). In addition, the expression levels proteins related to the mitochondrial apoptotic pathway (Bax and Cytochrome c) were up-regulated by PTE treatment, indicating that this apoptotic pathway was activated (Figure 4A).

Mentions:
The Western blot and immunofluorescence results showed that PTE increased the NICD protein level in A549 cells (Figure 4A and 4B). We then determined if the increase in the NICD protein level could augment Notch1 signaling by measuring the activity of a downstream target, Hes1. As observed for the NICD protein, PTE induced Hes1 expression (Figure 4A and 4C). We then examined the possible mechanism underlying the induction of NICD expression by PTE treatment. NICD is produced when the Notch1 receptor is cleaved by the gamma secretase complex, which is composed of four subunits (Presenilin-1, Nicastrin, anterior pharynx-defective 1 (Aph-1) and presenilin enhancer 2 (Pen-2)) [11], [12]. Therefore, PTE treatment induced NICD protein expression at least partially by increasing the gamma secretase activity. The treatment of A549 cells with PTE induced the expression of two of the subunits, Presenilin-1 and Nicastrin (Figure 4A). In addition, the expression levels proteins related to the mitochondrial apoptotic pathway (Bax and Cytochrome c) were up-regulated by PTE treatment, indicating that this apoptotic pathway was activated (Figure 4A).

Bottom Line:
PTE treatment resulted in a dose- and time-dependent decrease in the viability of A549 cells.DAPT (a gamma secretase inhibitor) and Notch1 siRNA prevented the induction of NICD and Hes1 activation by PTE treatment and sensitized the cells to PTE treatment.The down-regulation of Notch signaling also prevented the activation of pro-survival pathways (most notably the PI3K/Akt pathway) after PTE treatment.

ABSTRACTAlthough pterostilbene (PTE) has been shown to have potent antitumor activities against various cancer types, the molecular mechanisms of these activities remain unclear. In this study, we investigated the antitumor activity of PTE against human lung adenocarcinoma in vitro and in vivo and explored the role of the Notch1 signaling pathway in this process. PTE treatment resulted in a dose- and time-dependent decrease in the viability of A549 cells. Additionally, PTE exhibited strong antitumor activity, as evidenced not only by a reduced mitochondrial membrane potential (MMP) and a decreased intracellular glutathione content but also by increases in the apoptotic index and the level of reactive oxygen species (ROS). Furthermore, PTE treatment induced the activation of the Notch1 Intracellular Domain (NICD) protein and activated Hes1. DAPT (a gamma secretase inhibitor) and Notch1 siRNA prevented the induction of NICD and Hes1 activation by PTE treatment and sensitized the cells to PTE treatment. The down-regulation of Notch signaling also prevented the activation of pro-survival pathways (most notably the PI3K/Akt pathway) after PTE treatment. In summary, lung adenocarcinoma cells may enhance Notch1 activation as a protective mechanism in response to PTE treatment. Combining a gamma secretase inhibitor with PTE treatment may represent a novel approach for treating lung adenocarcinoma by inhibiting the survival pathways of cancer cells.